Direct Photolabeling with [³²P]UDP-Glucose for Identification of a Subunit of Cotton Fiber Callose Synthase

Abstract

We have identified a 52 kilodalton polypeptide as being a likely candidate for the catalytic subunit of the UDP-glucose: (1-->3)-beta-glucan (callose) synthase of developing fibers of Gossypium hirsutum (cotton). Such a polypeptide migrates coincident with callose synthase during glycerol gradient centrifugation in the presence of EDTA, and can be directly photolabeled with the radioactive substrate, alpha-[(32)P]UDP-glucose. Interaction with the labeled probe requires Ca(2+), a specific activator of callose synthase which is known to lower the K(m) of higher plant callose synthases for the substrate UDP-glucose. Using this probe and several other related ones, several other proteins which interact with UDP-glucose were also identified, but none satisfied all of the above criteria for being components of the callose synthase.